Based upon their embryonic expression domains and the preliminary results from the Principal Investigator's gene knockout studies presented below, the Dlx5 and Dlx6 homeobox genes are major directors of morphogenesis and cellular differentiation of the fetal skeleton. Dlx5 and Dlx6 also fall within the candidate critical region for the human split hand/split foot developmental disorder (OMIM 183600), also termed ectrodactyly (Caldwell, 1996; Crackower et al., 1996). As demonstrated below, the Principal Investigator has already made a simultaneous knockout of both Dlx5 and Dlx6 (Dlx5/6). Both genes are located a few kb apart on the chromosome, and a targeted deletion of both genes was deleted simultaneously, since the combined Dlx5 and Dlx6 null mouse cannot be achieved by simple intermating of the individual Dlx5 and Dlx6 knockouts, as the two genes are too closely linked. The limb defects observed closely recapitulate the phenotype of the human split hand/split foot inherited human malformation. Dr. Bruce Gelb, IRPG project 3, is pursuing the human aspect of this work. The remaining Dlx5/6 null phenotype shows a severe dysmorphology of craniofacial, axial and appendicular skeleton. What is unclear at this point, and is the primary focus of this proposal (IRPG project 4) is the following: 1) what the individual contribution of Dlx5 and Dlx65 is to this striking phenotype affecting each major subdivision of the fetal skeleton; 2) what the developmental mode of action is of these two genes; 3) what their position is within the hierarchy of skeletal patterning genes; and 4) what their mechanism is of spatiotemporal embryonic transcriptional regulation.